The overall objective of this proposal is to understand the role of orbital fibroblasts in the pathogenesis of thyroid-associated ophthalmopathy (TAO), the ocular manifestation of Graves' disease (GD). In its most severe forms, TAO can threaten sight. There are currently no specific and effective therapies for TAO. Thus devising new treatments for TAO represents an important unmet need. Central components of TAO are localized inflammation and abnormal deposition of hyaluronan (HA). Cytokines produced by infiltrating T cells and orbital fibroblasts are thought to drive the pathogenesis of TAO. HA is synthesized by a family of three hyaluronan synthase (HAS) isoforms. Short chain HA, a product of multiple HAS isoforms, is highly inflammatory. CD34+ fibrocytes are circulating bone marrow derived hematopoietic stem cells. They express the chemokine receptor, CXCR4 and migrate to tissue injury in response to CXCL12. We have found that TAO orbital tissue comprises two discrete cell populations, including CD34+ and CD34- fibroblasts. Moreover, fibrocytes are recruited uniquely to the TAO orbit where they appear to give rise to CD34+ fibroblasts. In contrast, healthy orbital tissue contains only CD34- fibroblasts. Unexpectedly we found that fibrocytes and orbit-infiltrating CD34+ fibroblasts express thyrotropin receptor (TSHR), the central autoantigen in GD. When activated by GD-specific thyroid stimulating immunoglobulins (TSI) or with TSH, fibrocytes express high levels of inflammatory cytokines that are implicated in the pathogenesis of TAO. We have generated a new conceptual model of TAO based on two testable hypotheses. Hypothesis 1: recruitment of fibrocytes to the TAO orbit accounts for the two divergent populations of CD34+ and CD34- fibroblasts. CD34+ fibroblasts derive from fibrocyte precursors. They express TSHR and respond to TSH and TSI, accounting for the cytokines that drive TAO. On the other hand, CD34- fibroblasts over-express CXCL12 in TAO and thus orchestrate CD34+ fibrocyte recruitment to the orbit. Hypothesis 2: TAO orbit-infiltrating CD34+ fibroblasts uniquely express HAS1 enzyme and produce excessive highly inflammatory short-chain HA. To test these hypotheses, we now propose the following specific aims. Specific Aim 1: determine the mechanisms underlying TSHR-mediated cytokine production in fibrocytes and in orbit-infiltrating CD34+ fibroblasts and determine the mechanism for CXCL12 over-expression in TAO orbital tissue that may underlie fibrocyte recruitment. Specific Aim 2: define the mechanisms involved in the unique anatomic-site and disease-specific expression and induction by IL-1 of HAS1 and the production of short-chain HA by TAO orbital fibroblasts. Our results, we believe, support a new and potentially paradigm-shifting model for TAO. The proposed studies represent logical extensions of our preliminary findings. They should directly impact the field of TAO by defining potentially attractive therapeutic targets which can be tested in vitro and can be translated into clinical trials involving patients with this vexing disease.